Hepatitis A virus (HAV) is a picornavirus with a single-stranded RNA genome of approximately 7500 nucleotides. The wild-type strain of HAV grows poorly in cell culture, is not cytopathic, and yields a low titer of virus. A cell-culture adapted mutant has been isolated which grows significantly more efficiently in cell-culture and which is attenuated for marmosets and chimpanzees. The objectives of this project are to determine the genetic basis for virulence and adaptation to cell culture of HAV. 1. The importance of the 2B and 2C regions of the HAV genome for accelerated growth in vitro has been confirmed while a secondary role for the P3 region has been established. 2. New mutations have been identified in the 5' non-coding region which play a role in growth of a vaccine strain of HAV in a human lung cell line. 3. Infection of marmosets with chimeric viruses showed that multiple mutations are required for attenuation of virulence. 4. Two chimeric cDNA clones with genomic regions of the simian strain of HAV in the HM-175 strain background produced viable virus after transfection of cultured cells. 5. Clonal isolates of CV-1 cells were shown to differ greatly in their capacity to support HAV replication in vitro.